Bacterial pathogens are a significant cause of economic loss in commercial farm operations as well as a health issue in a wide range of animal populations, including humans.
Three of the most common bacterial pathogens involved in veterinary diseases are Mannheimia (Pasteurella) haemolytica (Mh), Actinobacillus pleuropneumoniae (Ap) and Pasteurella multocida (Pm). MA is primarily a bovine and ovine pathogen, Ap is a porcine pathogen and Pm is a multi-species pathogen including poultry, cattle and swine, and is also the causative agent of dog and cat bite infections in humans. Taken together they cause diseases resulting in major economic losses to the animal farming industry.
Current practice involves using bacterins or crudely modified live strains as vaccines. Such approaches are not entirely satisfactory as effectiveness can be inadequate and there is a significant incidence of adverse effects.
There is currently no single vaccine available to provide cross-species protection against infections caused by these three species, Mh, Ap and Pm. It would be useful to have a vaccine against the pathogenic bacteria of concern. Vaccine approaches to combat diseases caused by Mh are still mainly based on bacterins and live attenuated strains. More recent versions of live vaccines incorporate secreted or extracted Mh antigens such as neuraminidase, leukotoxin, sialoglycoprotease, outer membrane and uncharacterised proteins. Leukotoxin has traditionally been the main sub-unit vaccine candidate, although immunization with a mutant strain expressing non-toxic leukotoxin was still partially virulent in a calf challenge model and leukotoxin in combination with capsular polysaccharide was unable to produce a protective immune response. In contrast, LPS has been shown to stabilize leukolytic activity and so a conjugate vaccine based on LPS and detoxified leukotoxin may offer promise [Li, J., and K. D. Clinkenbeard. 1999. Infect. Immun. 67: 2920-2927].
Current vaccine approaches to combat diseases caused by Ap are based on live attenuated strains as they contain the highly labile Apx toxins, which induce neutralizing antibodies required for protection. These Apx toxins form the basis for the major sub-unit vaccine against Ap but only seem to induce partial clinical protection. Adhesins including the core OS of LPS have been proposed as improved vaccine candidates [Van Overbeke I., et al. 2003. J. Vet. Med. B. Infect. Dis. Vet. Public Health. 50: 289-293]. Current vaccines to prevent Pm disease in pigs consist of toxoids and somatic antigens such as capsules and outer membrane proteins. Pm was first shown by Pasteur to induce fowl cholera in chickens and current strategies for protection against this disease utilize an attenuated Pm strain. In general however, this strategy is severely limited as the immune response remains serotype-restricted and fails to provide cross-protection against other serotypes. The LPS of Pm has however been shown to play a partial role in the immunity to infection.
Considerable evidence has accumulated indicating that LPS from each of these organisms may be a good candidate for subunit vaccine design. LPS has been shown to be both visible and a major antigenic determinant on the surface of Mh and mAbs raised to A1 LPS facilitated phagocytosis but not complement mediated killing in vitro [Wilson, C. F., et al. 1992. Vet. Microbiol. 31: 161-168].
In Pm ribosome-LPS vaccines protected chickens against fowl cholera disease from homologous Pm strains [Phillips, M., and R. B. Rimler. 1984. Am. J. Vet. Res. 45: 1785-1789]. Additionally, immunization with a LPS-protein complex provided 100% protection to mice when challenged with a homologous strain yet when separated the individual components of the complex afforded no protection. MAbs raised to LPS from Pm only afforded partial protection in a mouse model and although they were opsonophagocytic, were not bactericidal in the presence of complement. However in another study a mAb to Pm LPS completely protected mice against homologous challenge and was bactericidal [Wijewardana, T. G., et al. 1990. J. Med. Microbiol. 33: 217-222]. An anti-idiotype vaccine mimicking LPS from type A was protective in a mouse model when challenge was with homologous organisms.
In Ap, the LPS and more specifically the core region of the LPS has been implicated in the adhesive abilities of the bacterium. A conjugate vaccine of BSA linked to Ap serotype 1 LPS was protective against mice following homologous but not heterologous challenge and conjugates of both smooth and rough Ap LPS from serotype 5 and 1 suggested that the carbohydrate portion of the cell wall of Ap plays a significant role in the porcine immune response [Fenwick, B., and B. I. Osburn. 1986. Infect. Immun. 54: 583-586].
In this context the veterinary organisms of interest contain surface exposed carbohydrate moieties that can be considered as vaccine candidates. These carbohydrate moieties include LPS and capsular polysaccharides. Capsular polysaccharides are repeating units of several carbohydrate residues directly linked to the bacterial surface whereas LPS consists of three regions, a lipid A region that links the LPS molecule to the bacterial surface via fatty acid residues, a relatively conserved core oligosaccharide region which links the lipid A region to the third region, the variable polysaccharide antigen (O-antigen). The heterogeneity of the capsular and O-antigenic polysaccharides from strain to strain would ordinarily preclude them as economically viable vaccine candidates due to their ability to provide coverage only to homologous strains. Recent advances in molecular genetics, molecular structure analysis and immunochemistry have provided powerful tools, which have allowed us to identify carbohydrate structures as candidate vaccine antigens.
Lipopolysaccharide (LPS) is an essential and characteristic surface-exposed antigen of Mh, Ap and Pm. (As discussed above, the terms lipopolysaccharide and LPS as used herein encompass short chain lipopolysaccharide and lipooligosaccharide (LOS)). Pm strains express heterogeneous populations of low-molecular-weight LPS, which can exhibit extensive antigenic diversity among multiple oligosaccharide epitopes, whereas strains of Mh and Ap produce both low-molecular-weight LPS and traditional LPS molecules with O-antigenic polymers. The LPS carbohydrate structures of Mh, Ap and Pm described by the Applicant herein can provide a source of protective antigens when they are presented to the host immune system in an appropriate fashion, for example, as a protein-conjugate. LPS proved useful as a vaccine candidate in Applicant's study because unexpectedly surface expressed carbohydrate antigens were identified which possess oligosaccharide epitopes that are genetically and physiologically stable, that are conserved across the range of strains, and that are accessible to host clearance mechanisms across the three species Mh, Ap or Pm.
The carbohydrate regions of Mh, Ap and Pm LPS molecules provide targets for recognition by host immune responses. Determination of structure is crucial to understanding the biology of Mh, Ap and Pm LPS and its role in bacterial virulence. Mh, Ap and Pm LPS comprises a heterogeneous mixture of molecules consisting of a variable oligosaccharide moiety and a membrane anchoring-Lipid A component and in the case of some strains of Mh and Ap a polymeric O-antigen. Based on the experiments described herein, a structural model was developed for Mh, Ap and Pm LPS consisting of a conserved tetra-heptosyl-di-glucosyl inner-core moiety, which is attached via a phosphorylated ketodeoxyoctonoate residue (Kdo) to a lipid A component and which has been found to be conserved and present in every strain so far examined.
From the structural analyses it was clear that the maximum structure absolutely conserved across the three species was that illustrated in Structure I below.
where Kdo is 3-deoxy-D-manno-2-octulosonic acid, Hep is heptose, Glc is glucose, P is phosphate and Lipid A is detoxified.
In order to utilize an antigen for vaccine development, four essential criteria must be fulfilled. That is the immunogenic epitope of the candidate antigen must be:
i) genetically stable;
ii) conserved in all clinically relevant strains across the species
iii) accessible (in vitro and in vivo) to host immune mechanisms; and,
iv) able to induce protective antibodies in vivo.
This invention has identified a conserved LPS carbohydrate epitope that has been shown to satisfy the majority of these criteria.
i) Genetic stability: The genes involved in the biosynthesis of the conserved inner core oligosaccharide have been identified in the genome strains of the three bacterial species, (two Pm strains, one Mh strain and two Ap strains). Genes involved in the different outer core variations exhibited by these bacterial species have been found to be variably present, and these data have been corroborated with structural data on these genome strains and additional strains of each bacterial species. However, both structural and genetic analyses (see Table A) have indicated that the inner core structure is structurally conserved due to the consistent presence of the genes known to be responsible for inner core biosynthesis.
TABLE AGlycosyltransferases of the conserved inner core LPS of the veterinarypathogens GenePm-3480Pm70Ap_1Ap_5MhkdtAContig49 e−155*PM1305 e−155Contig25 e−129ap93i1204 e−128Contig164 e−126opsXContig49 e−145PM1302 e−152Contig24 e−111ap93i0300 e−108Contig165 e−116rfaFContig68 e−165PM1844 e−165Contig25 e−153ap93i0451 e−154Contig81 e−155orfHContig28 e−132PM1294 e−132Contig24 e−83ap93i0686 e−120Contig109 e−132lgtFContig49 e−110PM1306 e−110Contig24 e−110ap93i0684 e−114Contig109 e−116lbgBContig59 e−infPM1144 e−infContig29 e−98ap93i1369 e−98Contig147 e−89*e-values based on Blasting veterinary pathogen genomes with inner core LPS glycosyltransferases from Haemophilus influenzae strain KW-20 (Rd), except lbgB where PM1144 from Pm70 genome was used.Genome data obtained from: Baylor College of Medicine, Houston (Mh); Department of Microbiology and Immunology, Laboratory for Genomics and Bioinformatics, Oklahoma University Health Sciences Center (Pm-3480 and Ap1); Institute for Biological Sciences, National Research Council, Ottawa, Canada (Ap5); Computational Biology Centre, University of Minnesota (Pm70).ii) Structural conservation: In every strain investigated by Applicant to date this tetra-heptosyl-di-glucosyl moiety has been contained within the following structural element (Structure II):
where: —R is H or phosphoethanolamine (PEtn), P is phosphate, R′ and R″ are H or oligosaccharide chain extensions, preferably not including β-D-Galp-(1-7)-D-α-D-Hepp-(1-6), and R′″ is a variable O-antigen in Ap, Kdo is 3-deoxy-D-manno-2-octulosonic acid and Lipid A is detoxified.
TABLE BStructure of conserved and variable regions in the core oligosaccharides of theLPS from the veterinary pathogens Mannheimia haemolytica (Mh), Actinobacilluspleuropneumoniae (Ap) and Pasteurella multocida (Pm).Strain/SpeciesSerotypeR′R″RMhA1Hβ-D-Galp-(1-7)-D-α-D-HepVp-(1-HA8HD-α-D-HepVp-(1-HSH1217Hβ-D-Galp-(1-7)-D-α-D-HepVp-(1-HAp1(1S)-GalaNAc-(1-4,6)-α-D-Gal-HH(1-3)-β-D-Gal-(1-2β-D-Glc-(1-D-α-D-HepV-(1-H5aHD-α-D-HepV-(1-H5bHD-α-D-HepV-(1-HPm*Pm70β-D-Glc-(1-α-D-GalpNAc-(1-3)-β-D-GalpNAc-PEtn(1-3)-α-D-Gal-(1-4)-β-D-Gal-(1-4)-β-D-Glc-(1-VP161PCho-3-β-D-Gal-(1-PCho-3-β-D-Gal-(1-HX73(PEtn-6)-PCho-3-β-D-Gal-(1-(PEtn-6)-PCho-3-β-D-Gal-(1-HFor Pm the heptose residue, HepIV is of the L-α-D configuration. Also in Pm, the α-glucose residue at the 6-position of HepI, is absent in a minority of glycoforms, concomitant with a structural difference at the Kdo residue, when instead of P-R at the 4-position of Kdo there is a second Kdo residue.
The first heptose residue (HepI) is the only heptose residue from which core oligosaccharide extension has been shown to occur in all three species, Ap, Mh and Pm. Outer core extension elongates further from the HepIV residue. No core oligosaccharide extension has been observed from HepII or HepIII in any species. O-antigen extension in Ap serotype 5 was found to occur from the HepIII residue [St. Michael, F. et. al. 2004, Carbohydr. Res., 339: 1973-1984], the location of O-antigen attachment to the core OS has not been shown in Mh. No antigenic polymeric repeating units have been observed in Pm. Four genes have been shown to be involved in O-antigen biosynthesis in Ap serotype 1, including rfbP and rfbU, mutants of which result in Ap strains without O-antigen [Labrie, J. et al. 2002, J. Endotox. Res., 8: 27-38]. The inner core of all three species can be further substituted with a phosphoethanolamine residue, either at the 3-position of the HepII residue in Pm strain Pm70, or at the Kdo-P residue in all species Ap, Mh, and Pm. In Pm, a minor population of LPS glycoforms have been shown to elaborate two Kdo residues instead of the Kdo-P or Kdo-P-PEtn moiety, the presence of the second Kdo residue is concomitant with the loss of the inner core α-Glc residue. Outer core variation has been observed both within and between the three species of interest. In Ap serotype 1 the outer core extension has been completely structurally characterised by NMR and shown to contain the rarely encountered open-chain N-acetylgalactosamine structure terminal to the oligosaccharide extension of (1S)-GalaNAc-(1-4,6)-α-Gal-(1-3)-β-Gal. This structure has also been inferred from mass spectrometry in serotypes 9 and 11 also. In Ap serotype 2 the HepIV residue was found to be di-substituted at the 4-position with a β-Glc residue and at the 6-position with a D, D-α-HepV residue. In Ap serotype 5 only the D, D-α-HepV residue is substituting HepIV. A similar outer core extension to that found for Ap serotype 5 is observed in Mh, where the second D, D-α-HepV residue is itself substituted at the 7-position with a β-Gal residue. In Pm, studies on two serotype 1 strains, VP161 and X73 revealed an unusual outer core extension wherein the HepIV residue was symmetrically substituted at both the 4- and 6-positions with β-Gal residues that themselves were mono-substituted with phosphocholine moieties at the 3-positions, or elaborated in addition to the phosphocholine residues, phosphoethanolamine residues at the 6-positions of the β-Gal residues (X73). Another study on the genome sequenced strain Pm70 revealed di-substitution of the HepIV residue at the 4- and 6-positions once again. The 4-position was substituted by a β-glucose residue and at the 6-position with the unusual pentasaccharide α-GalNAc-(1-3)-β-GalNAc-(1-3)-α-Gal-(1-4)-β-Gal-(1-4)-β-Glc.
iii) Accessibility: Since this inner core structure is consistently expressed in these three species it was important to ascertain if epitopes of this inner core structure were accessible and conformationally conserved regardless of the variability in outer core, O-antigen and other cell surface structures. To achieve this the conserved structure needed to be elaborated as a terminal structure and to that end glycosyltransferases were targeted that would expose this structure. The availability of the complete genome sequence of Pm strain Pm70 in conjunction with thorough knowledge of this strains LPS structure (see Example 3), facilitated tentative identification of the glycosyltransferases responsible for the biosynthesis of the Pm70 LPS structure. Other genome sequences are also evolving for a second Pm strain (P-3480), two Ap strains (serotypes 1 and 5) and one Mh strain (serotype A1), and a Bioinformatics approach was utilised in order to identify target genes for mutagenesis. Mh strain A1 was selected for mutagenesis studies, as the amount of O-antigen present in this organism is low compared to Ap and therefore should not interfere with subsequent immunological studies. As illustrated below, candidate glycosyltransferases were identified in both a) Mh strain A1 and b) Ap serotype 1.

A homologue of the H. ducreyi lbgA galactosyltransferase gene [Tullius, M. V. et al 2002, Infect. Immun. 70: 2853-2861] had been identified in Ap located between two genes that were found to be involved in core oligosaccharide biosynthesis by mini-Tn10 transposon mutagenesis [Galarneau, C. et al 2000, Pathogenesis, 1: 253-264]. The adjacent gene in Ap, lbgB showed considerable homology to a D-glycero-D-manno-heptosyltransferase from Haemophilus ducreyi. Blast analysis of the Mh genome sequence (Baylor College, Houston) with the lbgB gene sequence from Ap, revealed two adjacent homologues to the D-glycero-D-manno-heptosyltransferase in the Mh genome sequence. The best lbgB homologue identified was postulated to be the D-glycero-D-manno-heptosyltransferase responsible for the addition of the first D-glycero-D-manno-heptose residue (HepIV) in the extension from the first L-glycero-D-manno-heptose residue (HepI), and we therefore postulated that the second homologue, which we termed losB, was the D-glycero-D-manno-heptosyltransferase responsible for the addition of the second D-glycero-D-manno-heptose residue (HepV).
Utilising standard molecular biological techniques, this gene was mutated and the resulting LPS derived from the mutant strain was structurally characterised to show that the targeted conserved inner core structure had been obtained (see Example 7). MAbs and pAbs were raised to this inner core LPS structure in order to examine the degree of conservation and accessibility of this structure across a range of strains of these veterinary pathogens. Polyclonal sera were obtained that were cross-reactive with both LPS and whole cells of each of the three species, Mh, Ap and Pm. Four mAbs (G3, G8, E8 and D8) were obtained from one fusion that were specific for an inner core conformation as it is presented on whole cells of Mh. Three mAbs (3-4, 3-5 and 3-16) were obtained from a subsequent fusion that were capable of cross-reacting with LPS from all of the strains of interest from Ap, Pm and Mh thus establishing the potential of a conserved cross-reactive LPS epitope shared between these three significant veterinary pathogens. Ascites fluid was raised from four of these mAbs (G3, G8, 3-5 and 3-16) and examined by whole cell ELISA which, revealed that the LPS epitopes recognised by these mAbs were accessible on the whole cells, thus demonstrating that the conserved inner core epitopes are accessible on the cell surface. Furthermore, mAbs G3 and G8 facilitated passive protection in a mouse model of Mh infection, illustrating accessibility to this inner core structure in vivo.
iv) Functional antibodies: Polyclonal sera containing antibodies cross-reactive with LPS of the species of interest, and mAbs G8 and G3 were able to facilitate complement-mediated lysis of Mh cells. Passive protection studies were performed in the well established mouse model of Mh infection, which revealed that provision of mAbs (G3 and G8) specific for this conserved inner core LPS structure in Mh were able to protect against disease. Finally a conjugate vaccine was produced linking the conserved carbohydrate structure to the carrier protein HSA, and mouse sera raised following immunization with this conjugate was found to be cross-reactive with LPS and whole cells from the three species of interest.